1. Field of the Invention
This invention is directed to methods for genetic transformation of algae, and in particular to conversion of obligate phototrophic organisms to recombinant organisms capable of heterotrophic growth.
2. Review of Related Art
Photosynthetic algae are the primary producers in aquatic environments, accounting for a significant proportion of worldwide O2 production and CO2 fixation in aquatic environments. Tréquer, P., Nelson, D. M., Van Bennekom, A. J., DeMaster, D. J., Leynaert, A. & Quéquiner, B. 1995. “The silica balance in the world ocean: a reestimate,” Science 269:375-79. Algae-are also needed for aquaculture and are used to produce many valuable products. For example, algae are used for the production of pigments (e.g., β-carotene, phycobiliproteins), oils with nutritional value (e.g., docosahexaenoic acid), and stable isotope-labeled biochemicals (e.g., 13C-glucose). Algae are also used as food for human and animal consumption.
In general, algae require light to drive photosynthesis for the production of the chemical energy required for cellular metabolism. Many are obligate phototrophs, meaning they have an absolute requirement for light to survive. Droop MR “Heterotrophy of Carbon.” in Algal Physiology and Biochemistry, Botanical Monographs, 10: 530-559, ed. Stewart W D P, University of California Press, Berkeley (1974). Such algae are unable to utilize exogenous organic compounds (such as glucose, acetate, etc.) as an energy or carbon source. Some algae are able to utilize either internal or external fixed carbon. A small number of algae are obligate heterotrophs: they are incapable of photosynthesis, relying entirely on exogenous organic compounds as energy and carbon sources.
Large scale cultivation of photosynthetic algae requires a relatively controlled environment with a large input of light energy. The requirement for light and the high extinction coefficient of chlorophyll in these organisms has necessitated the design and development of novel systems for cultivation and large scale growth. Chaumont, D. 1993. “Biotechnology of algal biomass production: a review of systems for outdoor mass culture.” J. Appl. Phycol. 5:593-604. A common limitation to all of these systems is the need to supply light to the culture, making it advantageous to maximize the surface-to-volume ratio of the culture. As cell densities increase, self shading becomes a limiting factor of productivity, resulting in relatively low biomass levels.
Most commercial production techniques use large open ponds, taking advantage of natural sunlight, which is free. These systems have a relatively low surface area to volume ratio with corresponding low cell densities. It is also very difficult to exclude contaminating organisms in an open pond. This difficulty restricts the usefulness of open ponds to a limited number of algae that thrive in conditions not suitable for the growth of most organisms. For example, Dunaliella salina can be grown at very high salinities. Apt K E et al, “Commercial Developments in Microalgal Biotechnology,” J Phycol. 35:215-226 (1999).
Enclosed photobioreactors, such as tubular photobioreactors, are an alternative outdoor closed culture technology that utilize transparent tubes enclosing the culture minimizing contamination. They provide a very high surface to volume ratio, so cell densities are often much higher than those that can be achieved in a pond. However, even in technologically advanced photobioreactors, the maximum algal cell densities attained are relatively low. In both ponds and bioreactors, low densities necessitate large volume cultures, which can result in a substantial cost for harvesting the algae. Apt K E et al. (1999). Furthermore, all outdoor culture systems are subject to large variations in light intensity and temperature caused by diurnal and seasonal periodicity that makes maintaining maximal productivity and reproducibility problematic.
Numerous designs have also been constructed for the indoor, closed culture of algae using electric lights for illumination. Ratchford and Fallowfield (1992) “Performance of a flat plate, air lift reactor for the growth of high biomass algal cultures,” J Appl. Phycol. 4: 1-9; Wohlgeschaffen, G D et al. (1992) “Vat incubator with immersion core illumination—a new, inexpensive set up for mass phytoplankton culture,” J Appl. Phycol. 4:25-9; Iqbal, M et al. (1993) “A flat sided photobioreactor for culturing microalgae,” Aquacult. Eng. 12:183-90; Lee and Palsson (1994) “High-density algal photobioreactors; using light-emitting diodes,” Biotechnol. Bioeng. 44:1161-7. These systems are expensive to build and operate and are subject to the same surface-to-volume constraints and problems associated with low density yields as outdoor ponds. Apt K E et al. (1999).
The production costs of phototrophically grown diatoms and other microalgae are very expensive, resulting from low densities and high harvesting costs. Nevertheless for a small number of specific algal products this technology has proven very successful, producing many thousands of tons per year. Lee, Y-K (1997) “Commercial production of microalgae in the Asia-Pacific Tim,” J. Appl. Phycol. 9:403-11; Apt K E et al. (1999). Major products from photosynthetic microalgae include dried biomass or cell extracts from Chlorella, Dunaliella and Spirulina. These are primarily produced in large open ponds.
Growing algae heterotrophically in conventional fermentors is a potential alternative to ponds or photobioreactors and a potential means to reduce substantially the cost of growing algae. Day et al. (1991) “Development of an industrial scale process for the heterotrophic production of a micro-algal mollusk feed,” Bioresource Technol. 38:245-9; Orus et al. (1991) “Suitability of Chlorella vulgaris UAM 101 for heterotrophic biomass production,” Bioresour. Technol. 38:179-184; Barclay et al. (1994) “Heterotrophic production of long-chain omega-3 fatty acids utilizing algae and algae-like microorganisms,” J. Appl. Phycol. 6:123-9; Gladue and Maxey (1994) “Microalgal feeds for aquaculture,” J. Appl. Phycol. 6:131-141; Chen F., “High cell density culture of microalgae in heterotrophic growth,” Trends Biotechnol. 14:421-6 (1996); Apt, K E et al. (1999). The basic principle of fermentor growth is to provide highly controlled optimal growth conditions to maximize productivity.
Typical fermentor culture conditions may be summarized as follows. The culture vessels range in volume from 1 to 500,000 liters and are operated under sterile conditions. A motorized shaft with a series of impellers provides mixing. Sterile air is pumped into the system at high pressure and flow rates to ensure proper gas exchange, and dissolved O2 and CO2 levels are continuously monitored and adjusted. Heating and/or cooling coils regulate temperature and the automatic addition of acid and/or base maintains pH. The culture medium for algal fermentative growth is similar to that used for phototrophic growth, except that glucose or a similar carbohydrate provides both fixed carbon and an energy source in fermentative growth. Other nutrient levels (i.e., nitrogen and phosphorus) are also continuously monitored and adjusted. Culture density may be further increased by using techniques such as chemostat culture, fed-batch culture, or membrane bioreactor culture. More detailed information regarding the growth of microalgae in fermentors can be found in the papers cited herein, which are herein incorporated by reference, and in Apt, K E et al. (1999). See, also, U.S. Pat. No. 5,244,921 to Kyle et al.; U.S. Pat. No. 5,374,657 to Kyle; U.S. Pat. No. 5,550,156 to Kyle; U.S. Pat. No. 5,567,732 to Kyle; U.S. Pat. No. 5,492,938 to Kyle et al.; U.S. Pat. No. 5,407,957 to Kyle, et al.; U.S. Pat. No. 5,397,591 to Kyle et al.; U.S. Pat. No. 5,130,242 to Barclay; U.S. Pat. No. 5,658,767 to Kyle; and U.S. Pat. No. 5,711,983 to Kyle which are also incorporated by reference.
As a result of the high level of process control possible with heterotrophic growth in fermentors, culture conditions and biomass yields are consistent and reproducible, with heterotrophic algal cell densities reported of 50 grams dry biomass per liter to as high as 100 g dry biomass per liter. Gladue and Maxey (1994); Running et al. (1994) “Heterotrophic production of ascorbic acid by microalgae,” J. Appl. Phycol 6:99-104. These biomass levels are at least 10-fold higher than those achieved by photosynthesis-based culture systems. Radmer R J and Parker B C (1994) “Commercial application of algae: opportunities and constraints,” J. Appl. Phycol 6:93-98. The high biomass levels also greatly decrease the volume of water that must be processed during harvesting per gram of biomass yield. Because cultures can routinely run in fermentors with volumes greater than 100,000 L, several thousand kilograms of dried biomass can be produced per run. The effectiveness of large-scale cultures and the production of high biomass levels can make the cost of fermentative growth an order of magnitude less expensive than photobioreactors. (Radmer and Parker 1994; Apt K E et al (1999).
The ability to provide complete control over the culture is also critical for maintaining food industry standard Good Manufacturing Practices (GMP), as designated by the Food and Drug Administration. Maintenance of GMP is required for the production of high-quality food- or pharmaceutical-grade materials. Apt K E et al. (1999).
The ability to grow microalgae heterotrophically using microbiological fermentation techniques can dramatically lower the costs associated with their production and provide the high degree of quality control needed for a food grade product. The estimated cost of producing heterotrophic algal biomass can be less than $5 per kilogram. Gladue and Maxey (1994). In contrast, the theoretical cost of producing, algae phototrophically in bioreactors is estimated to be an order of magnitude higher and actual production costs for phototrophic algae at aquaculture facilities are often two orders of magnitude higher. Wilkinson, L. (1998) “Criteria for the design and evaluation of photobioreactors; for the production of microalgae,” World Aquaculture Society Annual Meeting, February, Los Vegas, Nev., p. 584; Benemann, J. R. (1992) “Microalgae aquaculture feeds,” J. Appl. Phycol. 4:232-45. Only a small number of algae are currently produced using fermentation technology; these include Chlorella, Nitzschia alba, Tetraselmis, and Crypthecodinium. These algae are able to utilize external organic compounds as energy and carbon sources.
Given the valuable products produced by algae (including algal biomass itself and the difficulties of culturing algae in photosynthetically-based systems, it is highly desirable to culture microalgae in heterotrophic conditions in fermentors. However, a significant restriction on the use of fermentation technology for the production of algal products is that most algae are obligate phototrophs and are therefore unable to be grown using this technology. There therefore exists a need to develop methods for culturing a greater variety of algae under heterotrophic conditions in fermentors.